STAT3 is persistently activated in over a dozen types of human cancers, including all the major carcinomas, including breast, brain, colon, pancreas, ovarian, and squamous cell carcinomas of head and neck (SCCHN) cancers, and melanomas as well as some hematologic tumors (Bowman T, et al (2000) Oncogene 19, 2474-88, and Darnell, J. E. (2005) Nat. Med. 1 1, 595-596). As such, there is increasing interest in developing anticancer therapies through the inhibition of persistently active STAT3, especially as a strategy to deal with cancers where physicians are looking to improve the outcome and/or where even establishing a satisfactory standard of care has been challenging in terms of patient care, quality of life and outcome.
Glioblastoma (GBM) is considered the most aggressive and lethal of brain cancers, with a median survival after treatment of approximately 15 months. Shockingly, these modest results can only be achieved in the relatively young (i.e., <age 70) and otherwise healthy patients. Older patients with GBM, of which there are many, and those with poor performance status at diagnosis have much shorter survivals following identical therapy. In addition, GBM is occurring with increasing frequency in an aging population. Moreover, unlike the more common cancers, such as those of the lung, breast and colon, GBM is neither preventable, nor detectable at a stage when early treatment might be expected to be substantially more effective. Furthermore, despite decades of intensive research, major improvements in overall survival have remained elusive. As such, the development of therapeutic approaches to meet this unmet need is critical.
Brain tumours have been demonstrated to contain rare subpopulations of brain tumour stem cells (BTSCs), which possess the cardinal stem cell properties of clonogenic self-renewal, multipotency and tumourigenicity. The extensive self-renewal and proliferative capacity of BTSCs coupled with their insensitivity to conventional radio- and chemotherapies suggest that they are integral to the growth and post-treatment recurrence of GBM. As such, BTSCs represent a “reservoir of disease” that require novel therapeutic approaches to effectively eliminate in order to improve the outcome of GBM.
STAT proteins were originally discovered as latent cytoplasmic transcription factors that mediate cytokine and growth factor responses (Darnell, J. E., Jr. (1996) Recent Prog. Norm. Res. 51, 391-403; Darnell, J. E. (2005) Nat. Med. 1 1, 595-596). Seven members of the family, STAT1, STAT2, STAT3, STAT4, STAT5a and STAT5b, and STATE, mediate several physiological effects including growth and differentiation, survival, development and inflammation. STATs are SH2 domain-containing proteins. Upon ligand binding to cytokine or growth factor receptors, STATs become phosphorylated on critical Tyr residue (Tyr705 for STAT3) by growth factor receptors, cytoplasmic Janus kinases (Jaks) or Src family kinases. Two phosphorylated and activated STAT monomers dimerize through reciprocal pTyr-SH2 domain interactions, translocate to the nucleus, and bind to specific DNA-response elements of target genes, thereby inducing gene transcription (Darnell, J. E., Jr. (1996) Recent Prog. Norm. Res. 51, 391-403; Darnell, J. E. (2005) Nat. Med. 1 1, 595-596). In contrast to normal STAT signaling, many human solid and hematological tumors harbor aberrant STAT3 activity (Turkson, J. Expert Opin. Ther. Targets 2004, 8, 409-422; Darnell, J. E., Jr. (1996) Recent Prog. Norm. Res. 51, 391-403; Darnell, J. E. (2005) Nat. Med. 11(6), 595-596; Bowman, T. et al. (2000) Oncogene 19(21), 2474-2488; Buettner, et al. (2002) Clin. Cancer Res. 8(4), 945-954; Yu, H. and Jove. R. (2004) Nat. Rev. Cancer 4(2), 97-105; Haura, E. B., et al. (2005) Nat. Clin. Pract. Oncol. 2(6), 315-324).
Of note, STAT3 protein is one of seven family members of the STAT family of transcription factor proteins. STAT3 is activated through phosphorylation of a tyrosine 705 (Y705) that initiates complexation of two phosphorylated STAT3 monomers (pSTAT3). pSTAT3 homodimers are mediated through reciprocal STAT3 Src Homology 2 (SH2) domain-pY705 STAT3 interactions. pSTAT3:pSTAT3 homodimers translocate to the nucleus and bind DNA, promoting STAT3 target gene transcription. Targeting STAT3 has been previously achieved with dominant negative constructs, oligonucleotides or, most commonly, phosphopeptidic agents that mimic the native pY705 containing binding sequence.
Unfortunately, these inhibitors are rapidly degraded in vivo, which limits their use in the clinic. To circumvent these problems, small molecule STAT3 inhibitors were designed for treatment of cancers harboring hyperactivated STAT3 protein. Acid-based inhibitors have been identified in WO2012/018868 that potently and selectively block STAT3 dimerization and DNA-binding activity, namely, compound 45O, also referred to as BP-1-102 (sometimes referred to as compound 1 herein). Compound 45O in WO2012018868 potently suppresses multiple oncogenic properties in diverse cultured cancer cells (breast, lung, pancreatic, prostate, lung), including: cell proliferation, anchorage-independent cell growth, migration, invasion and motility. It is selective for STAT3, with over 10-fold less binding to 93% homologous STAT protein, STAT1. It showed little or no effect on phosphorylation of Shc, Src, Jak-1/2, Erk1/2 or Akt and had no effect on non-transformed cells (NIH 3T3 cells, STAT3 null mouse embryo fibroblasts, or mouse thymus stromal cells, nor does it affect transformed cells that do not harbor activated STAT3). Moreover, BP-1-102 exhibited striking anti-tumor effects in vivo in murine xenograft models of lung or breast cancer resulting in dramatic regression in tumor volumes. Western blots of residual tumors from treated mice showed repression in pSTAT3, cMyc, Cyclin D1, Bcl-xL, Survivin, and VEGF in a dose-dependent manner.
Moreover, genetic and other molecular evidence reveals persistent Tyr phosphorylation of STAT3 is mediated by aberrant upstream Tyr kinases and shows cancer cell requirement for constitutively-active and dimerized STAT3 for tumor maintenance and progression. Thus, in numerous proof-of-concept studies (Turkson, J., et al. Mol. Cancer Ther. 2004, 3(3), 261-269; Turkson, J., et al. J. Biol. Chem. 2001, 276(48), 45443-45455; Siddiquee, K.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 7391-7396; Turkson, J.; et al. Mol. Cancer Ther. 2004, 3, 1533-1542; and Turkson, J.; et al. J. Biol. Chem. 2005, 280(38), 32979-32988), inhibition of STAT3 activation or disruption of dimerization induces cancer cell death and tumor regression. Small-molecule STAT3 inhibitors thus provide tools for probing the molecular dynamics of the cellular processing of STAT3 to understand STAT3's role as a signaling intermediate and a molecular mediator of the events leading to carcinogenesis and malignant progression. Moreover, since the STAT3 pathway is a key oncogenic driver in over a dozen types of human cancers, including all the major carcinomas, including breast, brain, colon, pancreas, ovarian, and squamous cell carcinomas of head and neck (SCCHN) cancers, and melanomas as well as some hematologic tumors (Bowman T, et al (2000) Oncogene 19, 2474-88, and Darnell, J. E. (2005) Nat. Med. 1 1, 595-596) the direct inhibition of STAT3 would provide a molecularly targeted route for effectively managing these cancers and especially aggressive forms such as GBM.
In a seminal paper, Carro et al. (Nature, 463(7279): 318-325, 2010) demonstrated that the Signal transducer and activator of transcription 3 (STAT3) gene abnormally active in GBM, is a critically important mediator of tumour growth and therapeutic resistance in GBM. Poorly treated brain cancers such as gliomas, astrocytomas and glioblastomas harbor constitutively activated STAT3. In addition, a growing body of recent evidence gathered using a variety of different small molecules that indirectly inhibit STAT3 by targeting upstream molecules such as the JAK family members, strongly suggest that STAT3 signaling is crucial for the survival and proliferation of BTSCs and GBM both in vitro and in vivo. However, due to their broad targeting nature existing drugs for treating GBM have limited translational potential due to numerous side effects. Hence, drugs with the ability to more specifically block STAT3 activity may provide effective treatment for GBM patients.
STAT5 signaling, like STAT3 signaling, is transiently activated in normal cells and is deactivated by a number of different cytosolic and nuclear regulators, including phosphatases, SOCS, PIAS, and proteasomal degradation. Like STAT3, STAT5 has gained notoriety for its aberrant role in human cancers and tumorigenesis, having been found to be constitutively activated in many cancers, including those of the breast, liver, prostate, blood, skin, head and neck. (Muller, J., et al. ChemBioChem 2008, 9, 723-727). In cancer cells, STAT5 is routinely constitutively phosphorylated which leads to the aberrant expression of STAT5 target genes resulting in malignant transformation. Cancer cells harbouring persistently activated STAT5 over express anti-apoptotic proteins, such as Bcl-xL, Myc and MCL-1, conferring significant resistance to natural apoptotic cues and administered chemotherapeutic agents. Of particular interest, STAT5 has been identified as a key regulator in the development and progression of acute myelogenic (AML) and acute lymphoblastic leukemias (ALL; Gouilleux-Gruart, V., et al. Leukemia and Lymphoma 1997, 28, 83-88; Gouilleux-Gruart, V., et al. Blood 1996, 87, 1692-1697; Weber-Nordt, R. M., et al. Blood 1996, 88, 809-816). Moreover, inhibitors of upstream STAT5 activators (such as JA and FLT3) have been shown to exhibit promising anti-cancer properties (Pardanani, A., et al. Leukemia 2011, 25, 218-225; Quintas-Cardama, A., et al. Nature Reviews Drug Discovery 2011, 10, 127-140).
It should be noted that, medical benefits through the inhibition of STAT3/5 are not limited to the various forms of cancer described herein where these targets are constitutively activated, but would also be applicable to treating other conditions where these pathways are know to play a key role, such as, but not limited to autoimmune disorders (Harris, T. J.; et al Immunol. (2007) 179(7): 4313-4317), inflammation associated with arthritis (Miyamoto. T, et al, Arthritis Research & Therapy (2012), 14(Suppl 1):P43), inflammatory bowel disease (IBD) (World J Gastroenterol. (2008) 14(33): 5110-5114.), diabetes (Mashili, F.; et al (2013) Diabetes 62(2), 457-465), irritable bowel syndrome (IBS); kidney disease (Weimbs, T., (2013) JAK-STAT, 2(2), 0-1) and organ transplant (Debonera, F.; et al (2001) J. Surg. Res. 96(2), 289-295).
Despite advances in drug discovery directed to identifying inhibitors of STAT protein activity, there is still a scarcity of compounds that are both potent, efficacious, and selective activators of STAT3 and STAT5 and also effective in the treatment of cancer and other diseases associated with dysfunction in STAT3, STAT5 or both proteins, and diseases in which one or both of STAT3 and STAT5 is involved. Moreover, there is still a need for optimization of potency and reduced pharmacokinetic liabilities of existing compounds. These needs and other needs are satisfied by the present invention.